Wnt10b protects cardiomyocytes against doxorubicin-induced cell death via MAPK modulation

Background Doxorubicin, an anthracycline chemotherapeutic known to incur heart damage, decreases heart function in up to 11% of patients. Recent investigations have implicated the Wnt signaling cascade as a key modulator of cardiac tissue repair after myocardial infarction. Wnt upregulation in murine models resulted in stimulation of angiogenesis and suppression of fibrosis after ischemic insult. However, the molecular mechanisms of Wnt in mitigating doxorubicin-induced cardiac insult require further investigation. Identifying cardioprotective mechanisms of Wnt is imperative to reducing debilitating cardiovascular adverse events in oncologic patients undergoing treatment. Methods Exposing human cardiomyocyte AC16 cells to varying concentrations of Wnt10b and DOX, we observed key metrics of cell viability. To assess the viability and apoptotic rates, we utilized MTT and TUNEL assays. We quantified cell and mitochondrial membrane stability via LDH release and JC-1 staining. To investigate how Wnt10b mitigates doxorubicin-induced apoptosis, we introduced pharmacologic inhibitors of key enzymes involved in apoptosis: FR180204 and SB203580, ERK1/2 and p38 inhibitors. Further, we quantified apoptotic executor enzymes, caspase 3/7, via immunofluorescence. Results AC16 cells exposed solely to doxorubicin were shrunken with distorted morphology. Cardioprotective effects of Wnt10b were demonstrated via a reduction in apoptosis, from 70.1% to 50.1%. LDH release was also reduced between doxorubicin and combination groups from 2.27-fold to 1.56-fold relative to the healthy AC16 control group. Mitochondrial membrane stability was increased from 0.67-fold in the doxorubicin group to 5.73 in co-treated groups relative to control. Apoptotic protein expression was stifled by Wnt10b, with caspase3/7 expression reduced from 2.4- to 1.3-fold, and both a 20% decrease in p38 and 40% increase in ERK1/2 activity. Conclusion Our data with the AC16 cell model demonstrates that Wnt10b provides defense mechanisms against doxorubicin-induced cardiotoxicity and apoptosis. Further, we explain a mechanism of this beneficial effect involving the mitochondria through simultaneous suppression of pro-apoptotic p38 and anti-apoptotic ERK1/2 activities.


Introduction
Doxorubicin (DOX) is an effective chemotherapeutic in the anticancer armamentarium [1].Used frequently against a broad range of neoplasms, the therapeutic reach of DOX is limited by serious cardiomyopathy, with a 9% incidence in the first year following treatment [2].Childhood cancer survivors have a 12.5% risk of developing heart failure in the 30 years following DOX treatment [2,3].These cardiotoxicities manifest in both the acute and chronic windows.Acute, within three days of administration, presents with chest pain, myopericarditis, dysrhythmias, and left ventricular dysfunction [4].Chronic issues, which can manifest up to 10 years after administration, lead to heart failure with an incidence of 1.7%.The prognosis of congestive heart failure secondary to doxorubicin cardiotoxicity is poor, with ~50% 1-year mortality [5].Apart from age, where youth and the elderly are at higher risk and have poor cardiovascular health, the main risk factor for cardiotoxicity is dosage, with rates exceeding 18% and 65% once dose reaches 350 and 550mg/m2, respectively [2].
While cancer affects up to one in three individuals, profound improvements in survival have revealed increased rates of cardiovascular disease resulting from cardiotoxic infliction of cancer treatments.Therefore, elucidating the pathological and potential protective molecular mechanisms in DOX-induced cardiomyopathy will provide the groundwork for beneficial therapeutic intervention against the cardiovascular sequelae of chemotherapy.The difficulty is discerning between intended anticancer, DNA intercalation and damage, and cardiotoxic adverse mechanisms.Several hypotheses have been explored.A few of note: DOX induces toxic mitochondrial iron accumulation, reactive oxygen species (ROS) formation via reduction by NADPH dehydrogenase, and DOX inactivation of pro-survival signaling proteins such as extracellular signal-regulated kinase (ERK) [2,3,6].
Recent investigations into molecular mitigation of ischemic cardiotoxicity observed that Wnt10b is strongly and transiently induced after myocardial infarction (MI) during the granulation tissue formation phase of cardiac repair, where neovascularization and fibrosis occur [7].The study found that Wnt10b overexpression in murine cardiomyocytes stimulates further blood vessel growth and suppression of fibrosis formation.Wnt10b belongs to the Wnt family of highly conserved molecules whose role in cardiovascular diseases has not been thoroughly investigated [8,9].
We investigated whether Wnt10b provides a cardioprotective role in the AC16 human cardiomyocytes by translating these findings from ischemia-induced to DOX cardiomyopathy.Our results demonstrate that Wnt10b protects DOX-induced cell damage, reduces plasma membrane and mitochondrial membrane damage, and mitigates apoptosis by modulation of MAPK signaling of ERK1/2 and p38.

Cell culture
AC16 Human Cardiomyocyte Cell Line was purchased from Sigma (Saint Louis, MO) and maintained in DMEM/F12 with 4.5 g/L glucose supplemented with 10% (v/v) fetal bovine serum (FBS).Cells were cultured to 80% confluence in a 100 mm dish at 37˚C with 5% CO2 before passage and seeding for experiments.
For induction of cell death and apoptosis, AC16 cells were exposed to different concentrations of DOX for various times as indicated, following co-treatment with Wnt10b.The dose of Wnt10b was chosen based on published literature.The 200 nM of 48 hours of DOX-treatment was chosen based on our experiment, as shown in Fig 1A.

MTT: Determination of cell viability
MTT assay was performed to evaluate cellular viability.AC16 cells were plated in 96-well plates for 24 hours before experiments.After incubation with the treatments mentioned above, cells were continually cultured for 4 hours under the same conditions in fresh media containing MTT (5 mg/mL).Subsequently, the formazan crystals were solubilized in dimethyl sulfoxide (DMSO), and absorbance was measured at a wavelength of 570 nm.

Quantifying apoptotic rates: TUNEL assay
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed using an in-situ apoptosis detection kit (Roche) according to the manufacturer's protocol.After fixing with 4% paraformaldehyde and staining with TUNEL dye, the TUNEL-positive cells were identified by Leica confocal microscope.Results were expressed as the proportion of TUNEL-positive cells to total cells counted.

Measurement of mitochondrial membrane potential (ψ): JC-1 staining
Tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Invitrogen) was used to determine the changes in mitochondrial transmembrane potential.JC-1 exhibits potential-dependent accumulation in mitochondria.In normal healthy cells, JC-1 accumulates and forms dimeric Jaggregates in the mitochondria, giving off a bright red fluorescence.However, when the potential is disturbed, the dye cannot access the transmembrane space and remains in the cytoplasm in its monomeric form (green fluorescence).
Grown in 8-well glass chambers, AC16 cells were cotreated with Wnt10b and DOX for 24 hours.The cells were incubated with JC-1 dye in serum-free media for 15 minutes at 37˚C.The media were then removed, and cells were washed three times using PBS.JC-1 fluorescence was measured to assess the emission shift from green (530 nm) to red (590 nm) in polarized mitochondria at 488 nm excitation.

LDH cytotoxicity assay: Assessment of plasma membrane stability
Lactate dehydrogenase (LDH) assays were performed to quantify plasma membrane permeability changes during apoptosis using the LDH cytotoxicity assay kit (Promega).After the proper treatment, the culture media of AC16 cells was used to quantify LDH release.

Immunoblotting
Whole-cell lysates were prepared in RIPA buffer supplemented with protease cocktail inhibitor mix (Roche).The following quantification by Bradford protein assay and mixing with 5X SDS loading buffer, the lysate was heated to 100˚C for 6 minutes.Samples were subjected to SDS-PAGE electrophoresis using Tris-glycerol buffer.Proteins from the gel were transferred onto the PVDF membrane (Millipore).Membranes were probed with antibodies specific to Bcl-XL, Bcl2, p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, cleaved Caspase 3, and β-Actin at 4˚C overnight.After primary antibody incubation, membranes were incubated with goat antirabbit secondary antibody (LI-COR) and were exposed to the LI-COR image system.

Cell amplification assay
In total, 1 x 10 5 of the AC16 cells were grown in 6-well plates with DMEM/F12 + 10%FBS growth media.After being treated with Wnt10b (200 ng/ml) and DOX (200 nM), the cell number of each well was measured with Invitrogen™ Countess™ II Automated Cell Counter at 12 h, 24 h, 36 h, and 48 h of time points.

Statistical analysis
Data were collected from experiments of each of the groups and are expressed as means + SEM.Statistical analysis was undertaken using Student's t-test.Differences between groups were considered significant at *p<0.05 and **p<0.01.

Cardioprotective effects of Wnt10b in AC16 cells
TUNEL staining and LDH release assays were used to elucidate any cardioprotective properties of Wnt10b in response to DOX toxicity.After 48 hours of exposure to 200 nM DOX, 70.1 ± 3.6% of AC16 cells demonstrated TUNEL-positive results, this reduced to 51.1 ± 4.36% in the cotreated Wnt10b + DOX group (n = 3, p<0.01)(Fig 2A and 2B).LDH assays were used to quantify further the protective effects on the plasma membrane by Wnt10b.AC16 cells co-treated with Wnt10b and DOX (1.56 ± 0.12 folds, n = 5, p<0.01) had a 30% reduction in LDH release relative to cells solely exposed to DOX (2.27 ± 0.1 folds, n = 5,p<0.01)(Fig 2C).

Wnt10b maintains mitochondrial membrane integrity via restoration of membrane potential (Δψm) and mitigates intrinsic apoptotic mechanisms during DOX exposure
JC-1 staining was used to evaluate if DOX-induced cardiotoxic stress triggers intrinsic cell death through the collapse of the mitochondrial membrane potential (Δψm).Using the ratio of JC-1 staining, with red (healthy) to green (damaged mitochondria) fluorescent cells, to quantify mitochondrial damage, when comparing mitochondrial damage after 200 nM DOX exposure, the Wnt10b pretreated group had a 5.73 ± 1.24-fold increase in the red-to-green ratio (n = 3, p<0.01)(Fig 3A and 3B).Exposing AC16 cells to varying concentrations of Wnt10b and DOX, we assessed the activity of critical modulators of apoptosis: caspase 3, Bcl2, and Bcl-xL.After exposure solely to 200 nM of DOX, cleaved caspase three levels increased 2.4 ± 0.11-fold relative to control.However, caspase three activity was attenuated to 1.3 ± 0.05-fold of control after exposure to 50nM of Wnt10b (n = 3, p<0.01)(Fig 3D).The expression Bcl class of anti-apoptotic proteins, while noted to be suppressed by DOX, was significantly increasingly induced by Wnt10b co-treatment.

MAPK signal finely modulated by Wnt10b as a protective mechanism against DOX-induced cytotoxicity
Immunoblotting was utilized to investigate the interaction of Wnt10b and DOX on the MAPK signaling cascade.As per Fig 4A and 4B, antibody probing revealed that the ratio of phosphorylated-ERK1/2 to ERK1/2 was strongly induced by exposure solely to increasing concentrations of Wnt10b.While DOX suppressed the pERK1/2 ratio, its induction was salvaged with escalating co-treatment of Wnt10b (1.11 ±0.07 folds of 50ng/ml Wnt10b+DOX, and 1.37 ± 0.11 folds of 200ng/ml Wnt10b + DOX respectively vs 0.89+0.12folds of DOX group).Meanwhile, the opposite trend was found with the ratio of phosphorylated p38 to p38.Exclusive Wnt10b exposure reduced phosphorylated p38 by 12% and 19% in 50 nM (0.88 ± 0.1 folds) and 200 nM (0.81 ±0.09 folds) groups, respectively.DOX strongly induced p38 by a 1.25 ± 0.1-fold relative to control (1 ± 0.04), with Wnt10b co-treatment strongly reducing its expression to levels below control.

Wnt10b protects against DOX-induced cell damage apoptosis via ERK1/2 and p38 MAPK signal regulation
Utilizing inhibitors of the MAPK signaling cascade-ERK1/2 inhibitor FR180204 and a p38 inhibitor SB203580 -in combination with variable concentrations of Wnt10b and DOX treatment with AC16 cells, we investigated the relationship between Wnt10b and MAPK signaling.TUNEL staining and LDH release assays were utilized to quantify the resultant cytotoxic effects of targeted MAPK signaling blockade.Conversely, the addition of p38 inhibitor SB203580 reduced the TUNEL-positive cell percentage relative to the cotreated Wnt10b and DOX AC16 cells from 50.3 ± 0.58% to 44.5 ± 2.8% (n = 4, p<0.01) (Fig 5B).These results were again paralleled by the LDH release assay, where there was a 18.3% reduction from the Wnt10b cotreated group (1.64 ±0.07) to the p38 inhibitor addition (1.34 ± 0.04) (n = 5, p<0.01) (Fig 5B).

Discussion
The primary findings of our study suggest 1) Wnt10b preserves cell viability in cardiomyocytes exposed to DOX. 2) Wnt10b engages and enhances MAPK signaling via ERK1/2 upregulation and p38 downregulation, resulting in cell preservation.3) Wnt10b preserves mitochondrial membrane stability and 4) attenuates intrinsic apoptotic signaling by bolstering pro-survival proteins, Bcl-2 and Bcl-xL.Doxorubicin (DOX) cardiotoxicity symptoms span a continuum: from subclinical reductions in left ventricular ejection fraction (LVEF) and a rise in biomarkers of cardiac damage to symptomatic congestive heart failure (CHF) and arrhythmias [2].The cardiovascular toxicity due to DOX is dose-dependent, with a reported incidence of CHF as high as 4.7%, 26%, and 48% at DOX doses of 400, 550, and 700mg/m2, respectively [10][11][12].Further, with cardiac biomarker rise occurring in up to 40% of patients after administration, the cardiotoxicity of DOX appears to steer cardiomyocyte fate toward apoptosis [13][14][15].While the causative mechanisms of cardiac injury have yet to be identified, various hypotheses have been proposed.Our results of human cardiomyocyte cell line exposed to increasing DOX dosage demonstrated visible deformation, plasma membrane rupture per increase in LDH released, and apoptotic cell death.
We investigated the interplay between DOX and MAPK signaling and the potential role Wnt10b had in rescuing cardiomyocytes under DOX-induced duress.The question of Wnt10b, a ligand triggering the canonical Wnt signaling pathway, as an agent against DOXcardiotoxicity was inspired by a demonstration of protective effects from ischemic insult.Hatzopoulos et al. found that Wnt10b, released from intercalated discs after myocardial infarction, promotes neoangiogenesis, reduces scar size, and preserves ventricular function.By cotreating human cardiomyocytes with DOX and Wnt10b, we found cell viability was preserved, with significant reductions in apoptosis and plasma membrane stability maintained.
Growing evidence of a Wnt-MAPK connection made it an appealing target of DOX cardiotoxicity investigation.The Wnt-MAPK interplay ranges from physiologic functions, such as chondrogenesis, to malignancy, of which note, pro-and anti-apoptotic proclivities are tumordependent [8,16,17].Further, anthracyclines demonstrate pathologic alteration of the Neuregulin-1 (NRG-1) cascade, a pathway upstream of MAPK [2].Proteins ErbB2 and ErbB4, downstream of NRG-1, have demonstrated mitigation of heart failure and, when activated to heterodimerize, trigger MAPK signaling [18].Cardioprotective ErbB2 and ErbB4 proteins are significantly disrupted by doxorubicin [19,20].Therefore, we investigated MAPK signaling proteins ERK1/2 and p38 to quantify molecular dysregulation secondary to doxorubicin.The apoptotic direction MAPK signaling pathways steer cells is cell type-dependent.Typically, p38 is proapoptotic, and ERK1/2 is protective [21].Our findings demonstrate progressive suppression of ERK1/2 and conversely increased p38 activity with increasing DOX dose.With co-treatment of the Wnt10b ligand, the effects of DOX were reversed, with ERK1/2 levels rebounded and p38 suppressed.To further isolate the resultant effects on these MAPK proteins from the DOX-Wnt10b tug-of-war, we added ERK1/2 inhibitor FR180204 and p38 MAPK inhibitor SB203580 to the cotreated AC16 cells.Selective inhibition of ERK1/2 rendered the cytoprotective effects of Wnt10b null.At the same time, inhibition of p38 resulted in inverted results, with significant improvement in combating apoptosis.These findings suggest Wnt10b provides cardioprotective support to cardiomyocytes under DOX duress via modulation of the MAPK cascade by upregulation of anti-apoptotic ERK1/2 and suppression of pro-apoptotic p38.
Doxorubicin-induced damage at the mitochondrial level has garnered significant interest.One avenue of investigation is the unintended intercalation of DOX with DNA and topoisomerase 2β (Top2β), rather than the target isoform found in proliferating and neoplastic cells, Top2α [22][23][24].The therapeutic binding to Top2α in malignant cells leads to apoptosis, in binding to Top2β-an isoform found in quiescent cells-the cardiomyocyte develops mitochondrial dysfunction and succumbs to apoptosis [2,22,25,26].Top2β knock-out murine models demonstrated protection against DOX-cardiotoxic mitochondrial demise [27].While the iron-chelator dexrazoxane, the only FDA-approved prevention of DOX-cardiotoxicity, has been shown to inhibit Top2β, investigations by Ichikawa et al. suggest they are independent targets and pathways for cardioprotection [6].Further, the group demonstrated that dexrazoxane reduces DOX-induced mitochondrial iron accumulation and reverses cardiotoxicity [6].
The bifurcation in a cell's fate of demise or survival often rests in the in Bcl-2 class of proand anti-apoptotic proteins.The anti-apoptotic, Bcl-2 and Bcl-xL keep the pro-apoptotic Bax and Bak from perforating the outer mitochondrial membrane via induction of transmembrane pores [28].Permeabilizing this membrane opens Pandora's box: the uncoupling of oxidative phosphorylation, which yields rampant reactive oxygen species, and the release of cytochrome c, allowing assembly of key cell executor: caspase-3, culminating in apoptosis [28,29].Via voltage-dependent staining, we observed that DOX critically disrupts the mitochondrial membrane potential (Δψm).However, the co-treatment with Wnt10b salvaged not only Δψm, but also the anti-apoptotic proteins Bcl-2 and Bcl-xL.Meanwhile, the expression of apoptotic executor protein caspase 3, which DOX markedly induced, was suppressed by Wnt10b co-treatment.These results suggest that Wnt10b modulates mitochondrial regulatory proteins to preserve cell viability.

Fig 1 .
Fig 1. DOX causes cell toxicity in AC16 cardiomyocyte cells.(A-B) Results of cell viability analysis of cardiomyocytes that were treated with DOX for 48 hours at the indicated dose.Scale bar = 100 μm.(C) LDH release after DOX treatment at indicated dose for 48 h.(D) Representative immunoblots for protein expression of MAPK kinase and the quantitation of ERK1/2 and P38 activities (E, F).Each data point represents the mean + SD derived from four independent experiments.Differences between groups were considered significant at *p<0.05 and **p<0.01.https://doi.org/10.1371/journal.pone.0277747.g001